: The broad, long-term objective of this program is to study inositol metabolism in the opportunistic encapsulated fungal pathogen Cryptococcus neoformans. C. neoformans is typically found in such inositol-rich environments as the central nervous system (CNS) of immunocompromised individuals, AIDS and cancer chemotherapy patients, and soil contaminated with avail excreta. Only a few yeast species can catabolize inositol, half of these are of the genes Cryptococcus. Inositol availability for catabolism is contingent on transport from the environment into the cell(s). Recent studies have established that C. neoformans synthesizes inositol from glucose-6-phosphate, and catabolizes inositol to glucuronic acid. Provision of extracellular inositol has been shown to induce inositol catabolism and repress inositol synthesis. Furthermore, the regulation of inositol-containing lipid synthesis was shown to be different than that of other yeast and may represent a novel mechanism facilitating balanced growth in the inositol-rich central nervous system of infected animal hosts. The aim of this project is the molecular genetic analysis of inositol transport. Preliminary data suggest that, similar to higher eukaryotes, there are two inositol transporters in C. neoformans. Only one transporter, with low-affinity for inositol, is evident in kinetic studies of this organism grown in high levels of inositol. The specific project proposed is the isolation of genomic and cDNA clones encoding the C. neoformans low-affinity inositol transporter.The three alternate approaches which will be employed to isolate these clones are: 1) RT-PCR amplification of RNA enriched for the C. neoformans low affinity inositol transporter and subsequent hybridization to clone banks; 2) isolation and characterization of C. neoformans mutant strains defective in low-affinity inositol transport and complementation of these strains with clone banks of C. neoformans genomic DNA; 3) generation of probes by differential display and subsequent hybridization to clone banks of genomic and cDNA. The identity of the cloned DNAs obtained by any of these approaches will be verified by the other two approaches. Each of these experimental approaches individually should yield substantive information on inositol metabolism in this organism.